This project investigates the mechanisms of initial resistance to infection and generation of protective immunity to bacteria. The immune response to Francisella tularensis strain LVS, a gram negative facultative intracellular bacterium, is being characterized in terms of the cell types involved and their products (cytokines and antibodies). Results to date have demonstrated that mice which survive sublethal subcutaneous (sc) or intradermal (id) infection are solidly immune to subsequent lethal intraperitoneal (ip) or intravenous (iv) infection. Initial resistance to id infection is dependent on the production of Interleukin12, tumor necrosis factor and interferon gamma by macrophages and natural killer cells, respectively, since scid mice that lack mature T or B lymphocytes are able to survive sc or id infection for about 20 days. Studies in athymic nu/nu mice further demonstrate that mature T cells are required for long term resolution of id infection and survival. Cytokine production by T cells appears to be a major protective mechanism involved in generation of secondary immunity. However, a minor role for protection by antibacterial antibodies, that is itself dependent on T cells for expression, has been observed. A new major mechanism of protection has also been characterized in murine responses to Francisella: very strong specific protective immunity to LVS develops in both normal and nu/nu mice, but not scid mice, within 2-3 days after id priming with LVS. These results implicate a cell type present in nu/nu mice but not in scid mice that is able to generate immunity rapidly. In fact, both in vivo depletion studies and transfer studies suggest a major role for B220+ cells that is independent of antibody production. The mechanism(s) of this rapid generation of immunity may have important implications for development of vaccines to be used during community outbreaks of diseases. GRANT-Z01BJ05003 This project investigates the ability of recombinant vaccinia and herpes virus vectors to serve as delivery vehicles for immunomodulatory cytokines. Subcutaneous (sc) inoculation of vaccinia virus results in significant virus infection and replication in the skin, but much less in other major organs; thus recombinant vaccinia viruses may be especially well suited for delivery of immunomodulatory cytokines to skin-related rumors. Therefore studies to date have focused on the ability of recombinant vaccinia virus expressing murine Interleukin 4 (VV1/IL4) to modulate the course of skin tumor development in experimental animals (inbred mice). Repeated weekly treatment with VV1/IL4 sc at the site of tumor cell inoculation completely ablated subsequent tumor formation. This modulation of tumor growth was dependent on the dose of virus used for treatment. It was further dependent on the presence of mature T lymphocytes in the host, since repeated treatment of athymic nu/nu mice with VV1/IL4 had no effect on tumor development. No symptomatic toxicites were observed. Interestingly, treatment with a control recombinant virus that expressed only a marker gene, beta-galactosidase (VV2/bgal), also delayed or prevented sc tumor development in up to 40% of mice. Results to date suggest that nonspecific activation of macrophages and tumor necrosis factor production by vaccinia virus infection is responsible for some of the anti-tumor effects, and that viral delivery of IL4 additionally activates host tumor specific T cells to effect complete control of tumor development. However, in these studies determination of localization of IL4 was problematic due to the presence of endogenous host IL4. Thus mice lacking interferon gamma (GKO knockout mice) willl be used to characterize vaccinia mediated delivery of interferon gamma, as well as to study the ability of virus-delivered IFN gamma to treat bacterial diseases. Studies to characterize the localization of recombinant herpes viruses, which are expected to preferentially infect skin and nervous tissues, are also underway.